Direct quantitation of fatty acids present in bacteria and fungi: Stability of the cyclopropane ring to chlorotrimethylsilane

Article abstract of DOI:10.1021/jf8000313

The stability of the cyclopropane ring and the fatty acid composition of microbial cells were determined using chlorotrimethylsilane as reagent with three different conditions 80°C for 1 h, 60°C for 1 h, and 60°C for 2 h. Chlorotrimethylsilane permits a simultaneous extraction and derivatization of fatty acids. A basic method was used as reference. The bacteria, Escherichia coli, Burkholderia cepacia, and Lactobacillus brevis, and fungi Aspergillus niger and Gibberella fujikuroi were used. The stability of the cyclopropane ring on acidic conditions was tested using the cyclopropanecarboxylic acid and a commercial mixture of bacteria fatty acid methyl esters (BAME). Fisher's least significant difference test showed significant differences among the methods. The method using chlorotrimethylsilane and 1-pentanol for 1 h at 80°C gave the best results in cyclopropane, hydroxyl, and total fatty acid recoveries. This procedure allows the fast and easy one-step direct extraction derivatization.

Full text of DOI:10.1021/jf8000313

J. Agric. Food Chem. 2008, 56, 4923–4927 4923
Direct Quantitation of Fatty Acids Present in Bacteria
and Fungi: Stability of the Cyclopropane Ring to
Chlorotrimethylsilane
,†
†
‡
†
´
`
JORDI ERAS,* ROBERT ORO, MERCE TORRES, AND RAMON CANELA
Department of Chemistry and Department of Food Technology, University of Lleida,
Rovira Roure 191, 25198-Lleida, Spain
The stability of the cyclopropane ring and the fatty acid composition of microbial cells were determined
using chlorotrimethylsilane as reagent with three different conditions 80 °C for 1 h, 60 °C for 1 h, and
60 °C for 2 h. Chlorotrimethylsilane permits a simultaneous extraction and derivatization of fatty acids.
A basic method was used as reference. The bacteria, Escherichia coli, Burkholderia cepacia, and
Lactobacillus brevis, and fungi Aspergillus niger and Gibberella fujikuroi were used. The stability of
the cyclopropane ring on acidic conditions was tested using the cyclopropanecarboxylic acid and a
commercial mixture of bacteria fatty acid methyl esters (BAME). Fisher’s least significant difference
test showed significant differences among the methods. The method using chlorotrimethylsilane and
1-pentanol for 1 h at 80 °C gave the best results in cyclopropane, hydroxyl, and total fatty acid
recoveries. This procedure allows the fast and easy one-step direct extraction derivatization.
KEYWORDS: Chlorotrimethylsilane; fatty acids; gas chromatography; Aspergillus niger; Burkholderia
cepacia; Escherichia coli; Gibberella fujikuroi; Lactobacillus brevis
INTRODUCTION
using gas-liquid chromatography coupled mass spectrometry
(GLC-MS) data (9). The same method was used to establish
the influence of these acids in solvent tolerance and the survival
of Pseudomonas putida after lyophilization (10). Fatty acid
composition analysis has also led to the discovery of new fatty
acids with possible new applications (11). Moreover, the effect
of fatty acids biosynthesis inhibitors can be monitored by fatty
acid composition studies (12).
The study of the fatty acid compositions of bacteria and fungi
is useful for taxonomic studies. The analysis of the lipids present
in bacteria, the pattern of fatty acid methyl esters (FAMEs),
and the presence of unusual membrane fatty acids have been
extensively used to identify Gram-negative and Gram-positive
bacteria, to differentiate species, and to differentiate strains in
subgroups (1, 2). The fatty acid profile also has potential use
as a bioprospecting tool to locate fungi capable of producing
high value lipids, i.e., gamma-linolenic acid and arachidonic
acid (3). Moreover, commercially available microbe libraries
based on gas-liquid chromatography (GLC) analysis of fatty
acid composition have been used extensively in microorganism
taxonomy when grown and when harvest conditions are well
established (4–6).
The analysis of fatty acids also helps to explain the physi-
ological and biological activities of microorganisms. The
analysis of endogenous cell fatty acid composition, phospho-
lipids, and other membrane lipids allows the selection of suitable
growth factors, to study enzymatic activity, and to establish
oxygen tolerance (7, 8). Among these fatty acids, those based
on cyclopropane have an important role as constituents of
membrane phospholipids. The role of these compounds in the
acid shock response in Salmonella enterica has been studied
Because the study of fatty acid composition in bacteria
and fungi presents some special features, standard analytical
methods have been adapted to accommodate these particular
requirements (13). To determine the fatty acid composition
in acylglycerides, it is usual to perform a lipid extraction.
Once extracted, the lipid sample should be derivatized before
its characterization. Finally, the fatty acid composition can
be determined by GLC or GLC-MS analysis (14, 15). Three
factors are decisive in obtaining the quantitative recovery of
each fatty acid: the extraction method, the derivatizing
reagent, and the reaction conditions (16, 17). Cyclopropane
acids, which are characteristic of some bacteria, are consid-
ered to be sensitive to strongly acidic conditions. Conse-
quently, basic catalysis is an alternative method to analyze
these acids (18). However, basic catalysis is not recom-
mended for the analysis of free fatty acids (19). Methanolic
HCl seems to satisfy both requirements. Consequently, it has
been proposed as a system to derivatize both free fatty acids
and cyclopropane acids (20, 21).
* Corresponding author. Tel: +34 973702589. Fax: + 34 973238264.
E-mail: eras@quimica.udl.cat.
† Department of Chemistry.
The aim of this study was to demonstrate the applicability
of chlorotrimethylsilane and 1-pentanol as transesterification
‡ Department of Food Technology.
10.1021/jf8000313 CCC: $40.75  2008 American Chemical Society
Published on Web 06/06/2008

4924 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Eras et al.
Table 1. Influence of the Reaction Conditions on the Percentage of
Cyclopropanecarboxylic Ester Recovereda
ously described method introducing few modifications in temperature
and time reaction (23). The following three different conditions were
used for each sample: mixture heated at 80 °C for 1 h (method B);
mixture heated at 60 °C for 1 h (method C); mixture heated at 60
°C for 2 h (method D). Butylated hydroxytoluene was added as
antioxidant, and all assays were carried out in quintuplicate.
Transesterification of a Commercial Mixture of BAMEs. BAME
mix (Supelco (Madrid, Spain) in methyl caproate (800 µL) was
transesterified to pentyl esters using method B. The sample was
analyzed by GLC-MS.
conditions
60 °C 1 h
60 °C 2 h
80 °C 1 h
P value
methyl ester
pentyl ester
82.1 ( 1.8%a
77.2 ( 0.6%b
83.3 ( 1.0%a
0.002
0.766
96.9 ( 4.3%a
94.7 ( 1.3%a
96.1 ( 0.6%a
a Different superscript letters (a,b) in the same row indicate significant differences
among methods at the 95% level of significance according to Fisher’s least
significant difference test.
The results were compared with those obtained with GLC-MS from
the remaining commercial mix (0.2 mL).
reagent and extracting solvent to the determination of fatty
acid composition in a variety of microorganisms. Moreover,
the behavior of cyclopropane fatty acids under these direct
derivatization conditions was also studied. Because bacteria
and fungi can generate different results, three bacteria and
two fungi were chosen for this purpose. A method, exten-
sively used in the analysis of FAC in microorganisms, based
on a pretreatment with N,N-dimethylformamide (DMF) and
methylation with methanolic sodium methoxide (21), was
used as the reference.
Free Fatty Acid Analysis (Method E). Triundecanin (1 mL of 500
µg/mL) chloroform solution was added to a previously weighed 15
mL reaction vial fitted with a poly(tetrafluoroethylene) (PTFE)-lined
cap. The solvent was evaporated using a dry nitrogen stream. The
sample (100 mg), butylated hydroxytoluene (2 mg), and 2 mL of N,N-
dimethylformamide were then added to each reaction vial. The reaction
vials were filled with nitrogen and heated on a stirring block at 100 °C
for 30 min. After cooling, 20 mg of sodium chloride and 1 mL of
3.5% hydrochloric acid were added to each vial. The vials were
vortexed, extracted with 2 mL of a hexane-diethyl ether (1:1) mixture,
and centrifuged at 822g for 20 min.
The upper layers were recovered and dried over anhydrous magne-
sium sulfate. Each sample was analyzed by GLC-flame ionization
detection (FID) and GLC-MS, using triundecanin as the IS. All assays
were carried out in quintuplicate.
MATERIALS AND METHODS
Bacteria and Fungi Strains. Culture and Harvest. Three bacterial
strains, Escherichia coli (NCTC 9001, UdLTA 1.107), Lactobacillus
breVis (CECT 216, UdLTA 1.41), andBurkholderia cepacia [Pseudomo-
nas cepacia] (CECT 322, UdLTA 1.150), and two fungal strains,
Gibberella fujikuroi (UdLTA 3.104) and Aspergillus niger (UdLTA
3.37), were obtained from the collection of the Microbiology Unit of
the Food Technology Department, Lleida University (UdLTA).
The microorganisms were cultured under various conditions to
obtain the necessary cell masses for analysis. Escherichia coli and
Burkholderia cepacia were incubated in TSB broth (tryprone, 17
g/L; papaic digest of soybean meal, 3 g/L; glucose, 2.5 g/L;
dipotassium phosphate, 2.5 g/L; and sodium chloride, 5 g/L at pH
7.3) for 48 h at 30 °C, Lactobacillus breVis in MRS broth (proteose
peptone, 10 g/L; meat extract, 8 g/L; yeast extract, 4 g/L; glucose,
20 g/L; sodium acetate, 5 g/L; triammonium citrate, 2 g/L;
magnesium sulfate, 0.2 g/L; manganese sulfate, 0.05 g/L; dipotas-
sium phosphate, 2.0 g/L; and polysorbate 80, 1 g/L, pH 5.7) for
48 h at 30 °C, and Gibberella fujikuroi and Aspergillus niger in
ME broth (malt extract, 20 g/L; peptone, 10 g/L; glucose, 20 g/L;
pH 5.6) for 5 days at 25 °C, with orbital agitation.
Chromatographic and Statistical Analysis. Quantitative analysis
of the methyl and pentyl esters from the bacteria and Aspergillus niger
samples was carried out using a ThermoQuest Trace 2000 series GC
with an FID detector. The analytical column was a 30 m × 0.25 mm
fused silica capillary coated with a 0.20 µm film of poly(80%
biscyanopropyl-20% cyanopropylphenyl siloxane) (SP-2330; Supelco,
Madrid, Spain). The oven temperature was programmed to 100 °C for
the first 5 min, then increased to 220 at 5 °C/min, and then held
isothermally at 220 °C for 4 min. A 1:20 split injection ratio was used.
Helium was used as the carrier gas at a constant flow of 1 mL/min.
The injection volume was 1 µL. The injection system and the FID
system were held at 270 °C.
The esters from Gibberella fujikuroi were analyzed using a Hewlett-
Packard 6890 GC with an FID detector and a 105 m × 0.25 mm fused
silica capillary column coated with a 0.20 µm film of poly(90%
biscyanopropyl-10% cyanopropylphenyl siloxane) (RTX 2330; Restek,
Teknokroma, Sant Cugat del Valles, Spain). The oven temperature was
set to 100 °C for the first 10 min and was then increased to 240 at 3
°C/min and held for 30 min. The injection parameters were a 1:20 split
injection ratio, H2 as the carrier gas at a constant flow of 2 mL/min,
and an injection volume of 1 µL. The injection system and the FID
system were held at 270 °C.
Free fatty acids were analyzed using a Hewlett-Packard 6890 with
an FID detector and a 30 m × 0.25 mm fused silica capillary column
coated with a 0.25 µm film of treated polyethylene glycol (BP-21; SGE,
CromLab S.L., Barcelona, Spain). The oven was programmed to
increase in temperature from 90 to 240 °C at 12 °C/min and was then
held isothermally at 240 °C for 12 min. A 1:20 split injection ratio
was used, with helium as the carrier gas at a flow of 1 mL/min. The
injection volume was 1 µL. The injection system and the FID system
were held at 260 °C.
Qualitative analysis was performed with GLC-MS using an Agilent
6890N GC coupled to a 5973 Mass Selective Detector. The analytical
column was an SP-2330, as described above. The chromatographic
parameters were identical to those used for the analysis of the bacteria
and Aspergillus niger.
Cell masses of bacteria were collected by centrifugation at 10000g
for 15 min, and freeze-dried. Fungi were harvested by vacuum filtration
using a Bu¨chner funnel and freeze-dried. Bacteria and fungi were stored
at -30 °C until lipid analysis.
Esterification of Cyclopropanecarboxylic Acid. A mixture of 1.5
mmol of cyclopropanecarboxylic acid, 50 mmol of either methanol or
1-pentanol, and 3.5 mmol of chlorotrimethylsilane were added to a
Teflon screw-cap reaction vial filled with nitrogen. The vials with the
reaction mixture were heated and magnetically stirred under three
different conditions: (a) at 60 °C for 1 h, (b) at 60 °C for 2 h, or (c) at
80 °C for 1 h. After cooling, the mixtures were diluted with diethyl
ether and neutralized with saturated sodium bicarbonate solution. The
organic solutions were dried over anhydrous magnesium sulfate, and
2 mL of tridecane solution (1 mg/mL in isooctane) was added as an
internal standard (IS). All of the experiments were carried out in
triplicate. Compound purity was assessed by thin layer chromatography
(TLC) and GLC-MS. Methyl and pentyl cyclopropanecarboxylate yields
were calculated by GLC using the IS.
Transesterification with Sodium Methoxide (Method A). The
procedure, developed in previous studies (21, 22), consists of the
pretreatment of samples with N,N-dimethylformamide, followed by
derivatization with sodium methoxide in methanol. Butylated hydroxy-
toluene was added as an antioxidant, and all assays were carried out in
quintuplicate.
The response factors of methyl esters of commercially available
saturated and unsaturated fatty acids were calculated under the same
chromatographic conditions as those indicated above. The response
factors of pentyl esters were also calculated from the corresponding
esters obtained from 1-pentanol and each commercially available fatty
acids (23).
Transesterification with Chlorotrimethylsilane. The samples were
derivatized, without previous pretreatment, according to the previ-

Fatty Acid Quantification in Microorganisms
J. Agric. Food Chem., Vol. 56, No. 13, 2008 4925
Table 2. Amounts of Fatty Acids in Bacterial Samples According to Different Methods
methoda (mg fatty acidb/g bacterial sample)
A
B
C
D
Ec
P value
Escherichia coli
0.2c
C12:0
C14:0
C15:0
C16:0
C16:1d
C17:0
9.10-C16:0∆e
C18:0
C18:1d
0.4a
0.6b
0.4d
0.1
0.2
<0.1
0.3 ( 0.1
<0.1
0.1
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.110
3.2 ( 0.1a
4.4 ( 0.1b
2.9 ( 0.2c
3.5 ( 0.1d
0.6a
1.1b
18.5 ( 0.4a
1.0a
22.6 ( 1.0b
1.9 ( 0.3b
0.4 ( 0.1b
16.0 ( 0.6a
0.3 ( 0.1a
12.9 ( 0.5a
<0.1
17.0 ( 1.0c
0.6c
20.3 ( 0.5d
0.7c
0.2a
0.2a
0.3b
16.0 ( 0.3a
14.1 ( 0.8b
16.3 ( 0.5a
0.2a
0.2b
0.2a
0.1
0.2 ( 0.1
12.3 ( 0.6a,b
11.9 ( 0.1b
12.4a,b
0.020
C18:2
9.10-C18:0∆f
3-OH-C14:0g
total fatty acids
11.4 ( 0.7a
63.9 ( 1.0a
11.5 ( 0.3a
1.7 ( 0.1a
73.5 ( 1.0b
10.0 ( 0.6b
0.3b
57.4 ( 1.8c
11.4 ( 0.3a
1.4c
67.0 ( 1.4d
0.1
0.1 ( 0.1
1.2 ( 0.3
<0.001
<0.001
<0.001
Burkholderia cepacia
1.2 ( 0.1c
14.4 ( 0.2c
9.4 ( 0.3c
3.8 ( 0.1b
0.9 ( 0.4a
13.0 ( 0.2c
0.3b
C14:0
C16:0
0.8 ( 0.1a
10.8 ( 0.6a
7.4 ( 0.2a
3.6 ( 0.1a,b
0.7a
0.4b
1.5 ( 0.2d
17.5 ( 0.7d
11.4 ( 0.5d
4.5 ( 0.2c
1.1 ( 0.1a
15.8 ( 0.3d
0.4b
1.4 ( 0.1b
1.2 ( 0.1d
56.7 ( 1.3b
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
19.1 ( 0.6b
10.1 ( 0.3b
3.4 ( 0.2a
2.0 ( 0.1b
17.5 ( 0.3b
0.4b
0.1
0.1
1.2 ( 0.3
C16:1d
9.10-C16:0∆f
C18:0
C18:1d
10.6 ( 0.4a
1.7 ( 1.0
0.4 ( 0.3
3.5 ( 1.7
C18:2
0.1a
9.10-C18:0∆f
3-OH-C14:0g
total fatty acids
1.2a
1.1a
1.2 ( 0.1c
0.4 ( 0.2c
44.5 ( 1.2c
<0.1a
1.2 ( 0.1b
35.3 ( 0.9a
55.2 ( 1.2b
Lactobacillus brevis
C14:0
C15:0
C16:0
0.7a
0.2b
0.1a
3.1a
0.2a
0.3b
<0.001
0.172
<0.001
0.647
0.2a
0.1a
3.3a
0.2a
3.8 ( 0.1b
3.5 ( 0.2c
0.2
C16:1d
C17:0
0.2a
<0.1
0.2a
9.10-C16:0∆f
C18:0
0.1a
0.1a
3.3 ( 0.1a
11.2 ( 0.2a
18.2 ( 0.2a
0.1a
<0.1b
0.2c
2.4 ( 0.1c
8.6 ( 0.2c
14.9 ( 0.3b
<0.1a,b
0.3c
2.9 ( 0.4c
9.6 ( 1.4d
17.0 ( 2.1a
0.013
<0.001
<0.001
<0.001
0.001
0.7 ( 0.1b
4.5 ( 0.1b
7.4 ( 0.1b
17.7 ( 0.4a
<0.1
0.1
<0.1
0.3 ( 0.1
C18:1d
9.10-C18:0∆f
total fatty acids
a Standard deviations not presented are less than 0.1. b Expressed as acylglycerides except in method E. c Expressed as free fatty acid. d Undifferentiated cis-trans
isomers. e 9,10-Methylenehexadecanoic acid. f 9,10-Methyleneoctadecanoic acid. g 3-Hydroxytetradecanoic acid. Different superscript letters (a,b,c,d) in the same row indicate
significant differences among methods at the 95% level of significance according to Fisher’s least significant difference test.
Table 3. Comparison of Bacterial Acid Pentyl Esters and Commercial
the neutralization step (24). However, the high recoveries
BAMEs after the Application of Method B
found in all cases show that the cyclopropane ring is stable
under all of the conditions tested.
fatty acid ratio
BAMEs
bacterial acid pentyl esters
% difference
Once the stability of the cyclopropane acids was established,
the total fat and fatty acid composition of the bacterial samples
were determined using each method. Pretreatment and deriva-
tization with the sodium methoxide (method A) was selected
as the reference method because it has shown good performance
in the recovery of cyclopropane fatty acid (21).
C18:0/9,10-C16:0∆
C18:0/9,10-C18:0∆
C18:0/C18 unsata
C18:0/3-OH-C14:0
C18:0/C16:0
1.123
1.064
0.361
1.277
1.067
1.221
1.145
0.345
1.447
0.976
8.7
7.6
4.4
13.1
8.5
a C18 unsat: cis-9-C18:1, trans-9-C18:1, cis-9,12-C18:2.
Table 2 shows the results obtained after the application of
each method to the three bacteria studied. The amount of
acylglycerides was measured as milligrams of total acylglyc-
erides per gram of sample for each bacterium. Fisher’s Least
Significant Difference test showed significant differences among
the methods at the 95% level of significance. Considering that
method B did not differ significantly from methods A and D in
L. breVis with this test, it is reasonable to assert that method B
gave the highest fatty acid concentrations with the three bacteria
assayed. Method C gave the lowest fatty acid concentrations.
The results of reference method A were not significantly
different from those of method B for L. breVis. However, the
fatty acid composition determined by this method was signifi-
cantly lower than the value obtained with method B for E. coli
and B. cepacia. These differences among the methods could be
caused by the conversion of free fatty acids to sodium salts
Microsoft Excel 2003 for Windows was used to process the data.
RESULTS AND DISCUSSION
To determine the stability of the cyclopropane ring when
chlorotrimethylsilane was used, commercially available cyclo-
propanecarboxylic acid was esterified to methyl and pentyl
esters. The reaction was carried out using the methods B, C,
and D.
Table 1 shows that all three methods allowed the esteri-
fication of carboxylic acid with both alcohols. However, the
recovery of the methyl ester was poorer than the recoveries
of the pentyl ester. These differences could be explained by
the higher polarity and lower boiling point of the methyl ester.
These parameters could affect the recovery of the esters in

4926 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Table 4. Total Amount of Fatty Acids in Fungal Samples
method (mg fatty acida/g fungal sample)
Eras et al.
canoic acid was determined using the chlorotrimethylsilane
methods, whereas the results of basic catalysis with methanolic
sodium methoxide were poor.
A
B
P value
Our results are consistent with those of several similar studies
using acid catalysis. Lewis et al. (16) showed that direct
derivatization with dry HCl/methanol was better in terms of
total fatty acid present than a combination of extraction and
HCl/methanol derivatization. Similar results were described
when seven different methods were compared to analyze fatty
acid composition in lyophilized lactic acid bacteria. In this case,
the best method was the direct transesterification with a dry
HCl/methanol solution (21). The same authors also used a
method consisting of pretreatment with N,N-dimethylformamide
followed by sodium methoxide/methanol transesterification.
However, this method gave poorer results than the direct HCl/
methanol method. Nikkila et al. (26) carried out a study similar
to our own work. They compared the usefulness of sulfuric acid
as the only catalyst in a direct derivatization in front of the
combined basic-acid method as reference. Time and temper-
ature were optimized. They reported that recoveries of cyclo-
propane acids were best when sulfuric acid was used under
optimized conditions.
Gibberella fujikuroi
C14:0
C16:0
0.8 ( 0.1a
29.3 ( 2.9a
10.5 ( 1.1a
34.4 ( 3.5a
33.2 ( 3.2a
1.3 ( 0.1a
0.7 ( 0.1a
0.6 ( 0.1a
0.9 ( 0.1a
111.7 ( 11.1a
1.4 ( 0.1b
43.8 ( 2.9b
15.7 ( 1.0b
43.9 ( 3.4b
41.5 ( 3.2b
1.5 ( 0.2a
1.1 ( 0.1b
1.0 ( 0.1b
1.8 ( 0.1b
151.7 ( 11.0b
<0.001
<0.001
<0.001
0.002
0.003
0.062
<0.001
<0.001
<0.001
<0.001
C18:0
C18:1b
C18:2b
C18:3
C20:0
C22:0
C24:0
total fatty acids
Aspergillus niger
C15:0
C16:0
0.1a
0.3b
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.056
3.5 ( 0.2a
6.0b
C17:0
9.10-C16:0∆c
C18:0
0.1a
0.1a
0.4b
0.4b
1.6 ( 0.1a
7.7 ( 0.2a
8.6 ( 0.2a
1.4a
2.4b
C18:1b
C18:2b
6.6 ( 0.3b
7.1 ( 0.3b
0.9b
C18:3 + C20:0d
total fatty acids
23.2 ( 0.7a
24.1 ( 0.6a
To confirm the applicability of the selected method, it was
applied to the derivatization of a commercial BAME mixture
of fatty esters from bacteria. A comparative assay was performed
by splitting the commercial sample.
a Expressed as total acylglycerides. b Undifferentiated cis-trans isomers. c 9,10-
Methylenehexadecanoic acid. d Undifferentiated. Different superscript letters (a,b)
in the same row indicate significant differences among methods at the 95% level
of significance according to Fisher’s least significant difference test.
Table 3 shows the octadecanoic acid (C18:0):fatty acid ratios
of the commercial BAMEs and bacterial acid pentyl esters
prepared from this bacterial fatty acid mix using method B. Fatty
acids were chosen according to their putative sensitivity to the
derivatization conditions. Differences in percentages are also
shown for each derivatized fatty acid.
After derivatization, small differences (<10%), calculated as
percentages, were observed for 9,10-C16:0∆, 9,10-C18:0∆,
C16:0, and the combined cis-9,12-octadecadienoate, cis-9-
octadecenoate, and trans-9-octadecenoate acids. 3-Hydroxytet-
radecanoic acid showed a slightly higher difference (13.1%).
This assay confirmed the stability of cyclopropane and hydroxyl
fatty acids when method B was used.
Finally, although the analysis of fatty acid composition is
more difficult in fungi than in bacteria (27), the applicability of
method B to fungi was studied. Gibberella fujikuroi and
Aspergillus niger were used, and method A was used as the
reference method. Table 4 shows the results obtained for the
fungi using methods A and B. The individual concentrations
of each fatty acid were expressed as milligrams of individual
fatty acid per gram of fungal sample. Again, Fisher’s least
significant difference test showed that the total amounts of
acylglycerides determined by method B, expressed as milligrams
of total acylglycerides per gram of fungal sample, were either
equivalent to or significantly higher than the values determined
using reference method A.
As for most of the bacteria assayed, total fatty acids in G.
fujikuroi was higher with the chlorotrimethylsilane method than
with the sodium methoxide one. Both methods gave closer
results when applied to A. niger, with C16:0 acid being the only
exception. The significant difference between the two methods
in G. fujikoroi could be attributable to the higher levels of
saturated fatty acids in this fungus. In all cases, the recovery of
saturated fatty acids was greater when method B was used. The
recoveries of monounsaturated fatty acids and polyunsaturated
fatty acids were similar.
when the sodium methoxide method was used. However, when
the total free fatty acids were determined (Table 2), very low
amounts of free fatty acid were found in E. coli (1.2 ( 0.4
mg/g sample) and L. breVis. (0.3 ( 0.07 mg/g sample), whereas
B. cepacia had a higher amount (3.5 ( 0.1 mg/g sample).
Nevertheless, this amount represented less than 1% of the whole
amount of the acylglycerides present in B. cepacia. These results
indicate that free fatty acids are irrelevant in determining the
total acylglycerides in these samples. Therefore, it seems that
differences among the derivatization methods cannot be at-
tributed to the presence of free fatty acid in the microorganisms
studied but to the method used. It is more likely that these
differences result from the diversity of fatty acids present in
each microorganism and the conditions of each method tested.
The amount of each individual fatty acid was expressed as
milligrams of theorycal acylglyceride per gram of sample for
each bacterium (Table 2). Method B again produced the best
recovery of most of the saturated and unsaturated fatty acids,
whereas methods A and C gave the poorest recoveries.
Nevertheless, the percentages of 9,10-methylenehexadecanoic
acid and 9,10-methyleneoctadecanoic acid present in E. coli and
B. cepacia showed no significant difference among methods A
and B at the 95% level. For L. breVis, method A showed the
best recovery for 9,10-methyleneoctadecanoic acid. Method C
generally showed the poorest recovery. These results could be
attributable to the fact that method C is the shortest procedure,
considering its derivation period. Moreover, it is performed at
the lowest temperature. It seems that either a higher temperature
or extra time were required to efficiently extract and transform
the acylglycerides present in this bacteria.
Certain basic catalyses, used in several studies, are commonly
considered the best methods for bacterial fatty acid analysis (12).
It has been assumed that cyclopropane is more stable under basic
conditions than under acidic conditions (25). However, the three
chlorotrimethylsilane methods assayed allowed the determina-
tion of cyclopropane acids. The concentrations determined for
these acids were equivalent to or higher than those determined
using the reference basic method. Moreover, 3-hydroxytetrade-
We have demonstrated that chlorotrimethylsilane is a suitable
reagent for the direct analysis of the acylglycerides present in

Fatty Acid Quantification in Microorganisms
J. Agric. Food Chem., Vol. 56, No. 13, 2008 4927
inactivation of acyl carrier protein. FASEB J. 2006, 20, A849.
(13) Brondz, I. Development of fatty acid analysis by high-performance
liquid chromatography, gas chromatography, and related tech-
niques. Anal. Chim. Acta 2002, 465, 1–37.
(14) Meier, S.; Mjos, S. A.; Joensen, H.; Grahl-Nielsen, O. Validation
of a one-step extraction/methylation method for determination of
fatty acids and cholesterol in marine tissues. J. Chromatogr., A.
2006, 1104, 291–298.
(15) White, D. C.; Davis, W. M.; Nickels, J. S.; King, J. D.; Bobbie,
R. J. Determination of the sedimentary microbial biomass by
extractable lipid phosphate. Oecologia 1979, 40, 51–62.
(16) Young, J. C. Microwave-assisted extraction of the fungal me-
tabolite ergosterol and total fatty-acids. J. Agric. Food Chem. 1995,
43, 2904–2910.
(17) Lewis, T.; Nichols, P. D.; McMeekin, T. A. Evaluation of
extraction methods for recovery of fatty acids from lipid-producing
microheterotrophs. J. Microbiol. Meth. 2000, 43, 107–116.
(18) Christie, W. W. Preparation of ester derivatives of fatty acids for
chromatographic analysis. The Lipid Library. http://www.lipidli-
brary.co.uk/ (accessed April 29, 2007).
microorganisms. No extraction or pretreatment step is required,
and the recoveries are either similar to, or higher than, those
obtained with the sodium methoxide method. Method B involves
fewer steps than the sodium methoxide method. Consequently,
it allows the fast analysis of the total and individual fatty acids
present in bacteria and fungi. The whole period analysis could
be shortened using a rapid GC-FID method as recently proposed
(6). Moreover, the method developed here can be used to
identify and quantify the cyclopropane and hydroxy fatty acids
present in the microorganisms studied.
Considering some of our previous results (28, 29), several
alcohols can be used as the derivatizing agents. Consequently,
the method could be used with trichloroalcohols to determine
the presence of very low amounts of fatty acids in some
microorganisms (30). It is worth remembering that using
alcohols other than methanol improves the recovery of low-
molecular-weight fatty acids (26).
Supporting Information Available: Stoichiometry correction
factors and response factors relative to C11:0 for the chromato-
graphic assayed conditions. Examples of chromatograms of fatty
acids obtained with a ThermoQuest Trace 2000 series GC and
Hewlett-Packard 6890 GC. This material is available free of
charge via the Internet at http://pubs.acs.org.
(19) Liu, K. S. Preparation of fatty acid methyl esters for gas-
chromatographic analysis of lipids in biological materials. J. Am.
Oil Chem. Soc. 1994, 71, 1179–1187.
(20) Blomquist, G.; Andersson, B.; Andersson, K.; Brondz, I. Analysis
of fatty acids. A new method for characterization of moulds. J.
Microbiol. Meth. 1992, 16, 59–68.
(21) Dionisi, F.; Golay, P. A.; Elli, M.; Fay, L. B. Stability of
cyclopropane and conjugated linoleic acids during fatty acid
quantification in lactic acid bacteria. Lipids 1999, 34, 1107–1115.
(22) Suter, B.; Grob, K.; Pacciarelli, B. Determination of fat content
and fatty acid composition through 1-min transesterification in
the food sample; principles. Z. Lebensm.-Unters. Forsch. A 1997,
204, 252–258.
LITERATURE CITED
(1) Elvert, M.; Boetius, A.; Knittel, K.; Jorgensen, B. B. Characteriza-
tion of specific membrane fatty acids as chemotaxonomic markers
for sulfate-reducing bacteria involved in anaerobic oxidation of
methane. Geomicrobiol. J. 2003, 20, 403–419.
(23) Eras, J.; Ferran, X.; Perpin˜a, B.; Canela, R. Chlorotrimethylsilane,
a reagent for the quantitative analysis of fats and oils present in
vegetables and meat samples. J. Chromatogr., A. 2004, 1047, 157–
161.
(24) Nota, G.; Naviglio, D.; Romano, R.; Sabia, V.; Musso, S.
Evaluation and improvement of transesterification methods of
triglycerides. Anal. Lett. 1998, 31, 2499–2512.
(25) Moss, C., W New Methodology for the Identification of Nonfer-
menters: Gas-Liquid Chromatographic Chemotaxonomy. In Glu-
cose Non-Fermenting Gram-NegatiVe Bacteria in Clinical Mi-
crobiology; Gilardi, G. L., Ed. CRC Press: West Palm Beach,
FL, 1978; pp 171-201.
(26) Nikkila, P. A.; Johnsson, T. S. E.; Laakso, S. V. Direct acid-
catalysed transmethylation of lactobacillic acid in lactic acid
bacteria. Biotechnol. Tech. 1996, 10, 109–114.
(2) Mallory, L. M.; Sayler, G. S. Application of FAME (fatty-acid
methyl ester) analysis in the numerical taxonomic determination
of bacterial guild structure. Microb. Ecol. 1984, 10, 283–296.
(3) Kock, J. L. F.; Botha, A. Fatty Acids in Fungal Taxonomy. In
Chemical Fungal Taxonomy; Frisvad, J. C., Bridge, P. D., Arora,
D. K., Eds.; M. Dekker Inc.: New York, 1998; 219, p 246.
(4) Adams, D. J.; Gurr, S.; Hogge, J. Cellular fatty-acid analysis of
Bacillus thuringiensis var kurstaki commercial preparations. J.
Agric. Food Chem. 2005, 53, 512–517.
(5) Whittaker, P.; Fry, F. S.; Curtis, S. K.; Al-Khaldi, S. F.; Mossoba,
M. M.; Yurawecz, M. P.; Dunkel, V. C. Use of fatty acid profiles
to identify food-borne bacterial pathogens and aerobic endospore-
forming bacilli. J. Agric. Food Chem. 2005, 53, 3735–3742.
(6) Whittaker, P.; Keys, C. E.; Brown, E. W.; Fry, F. S. Differentiation
of Enterobacter sakazakii from closely related Enterobacter and
Citrobacter species using fatty acid profiles. J. Agric. Food Chem.
2007, 55, 4617–4623.
(27) Welch, D. F. Applications of cellular fatty-acid analysis. Clin.
Microbiol. ReV. 1991, 4, 422–438.
(7) Lee, Y. D.; Moon, B. Y.; Choi, J. P.; Chang, H. G.; Noh, B. S.;
Park, J. H. Isolation, identification, and characterization of aero-
adaptive Campylobacter jejuni. J. Microbiol. Biotechnol. 2005,
15, 992–1000.
(8) Partanen, L.; Marttinen, N.; Alatossava, T. Fats and fatty acids
as growth factors for Lactobacillus delbrueckii. Syst. Appl.
Microbiol. 2001, 24, 500–506.
(9) Kim, B. H.; Kim, S.; Kim, H. G.; Lee, J.; Lee, I. S.; Park, Y. K.
The formation of cyclopropane fatty acids in Salmonella enterica
serovar Typhimurium. Microbiol-SGM. 2005, 151, 209–218.
(10) Mun˜oz-Rojas, J.; Bernal, P.; Duque, E.; Godoy, P.; Segura, A.;
Ramos, J. L. Involvement of cyclopropane fatty acids in the
response of Pseudomonas putida KT2440 to freeze-drying. Appl.
EnViron. Microbiol. 2006, 72, 472–477.
(11) Zheng, C. J.; Yoo, J. S.; Lee, T. G.; Cho, H. Y.; Kim, Y. H.;
Kim, W. G. Fatty acid synthesis is a target for antibacterial activity
of unsaturated fatty acids. FEBS Lett. 2005, 579, 5157–5162.
(12) Rock, C. O. Inhibition of bacterial fatty acid synthesis via the
(28) Eras, J.; Montan˜es, F.; Ferran, X.; Canela, R. Chlorotrimethylsilane
as a reagent for gas chromatographic analysis of fats and oils.
J. Chromatogr., A. 2001, 918, 227–232.
(29) Eras, J.; Dolcet, A.; Ferran, X.; Canela, R. Chlorotrimethylsilane.
A reagent for the direct analysis of fats and paste samples. Food
Sci. Technol. Int. 2004, 10, 89–93.
(30) Brooks, J. Review of frequency-pulsed electron-capture gas-liquid
chromatography studies of diarrheal diseases caused by members
of the family Enterobacteriaceae, Clostridium difficile, and ro-
tavirus. J. Clin. Microbiol 1986, 24, 684–691.
Received for review January 4, 2008. Revised manuscript received
March 29, 2008. Accepted March 31, 2008. This work was supported
in part by a Grant-in-Aid from the Secretar´ıa de Estado de Pol´ıtica
Cient´ıfica y Tecnolo´gica of the Spanish Ministry of Education and
Culture (contract grant number: CTQ2006-07451/PPQ).
JF8000313